Evolution of IA pathology during 8-week time course of joint wound healing of FIX^−/−mouse following hemarthrosis. (A) Representative histopathology. Images were captured on a Nikon Microphor SA microscope equipped with 10/0.30, 20/0.50, and 40/0.70 numeric aperture objective lenses. Photographs were taken with a DMX-1200 color camera using the Act-1 software (entire system from Nikon Instruments, Melville, NY). Representative images of joint histology for each condition/time point are shown at original magnification ×40 (top) and ×200 (bottom). WT naïve and FIX^−/− naïve display normal histology of the uninjured mouse knee joint, with distal femur oriented on the left and proximal tibia on the right, with meniscus and synovial/subsynovial histology seen in the center, displaying normal 3-4 cell layer synovium overlying subsynovial fat with rare subsynovial blood vessels. Following hemarthrosis FIX^−/− mouse histology is remarkable on day 1 only for frank blood within the joint space and in the subsynovial space. Day 3 thickening of the synovial lining is evident. By day 7, the subsynovial space is replaced by a dense collection of inflammatory infiltrate and proliferative synoviocytes, which is punctuated by day 14 with frequent neovascular structures. Synovial hyperplasia resolves in some (although not all) FIX^−/− mice by day 56 following the single gross hemarthrosis; however, abnormal vascular changes persist. (B) Time course of synovial pathology. Joint capsule puncture was induced at day 0 in C57Bl/6J FIX^−/− mice, in 2 separate cohorts of C57Bl/6J hemostatically normal (WT) mice. NS was injected into the joint at the time of needle puncture for one-half of the WT mice (WT + NS, ▪) and for all of the FIX^−/− mice (FIX^−/−, ●). The other half of the WT mice received autologous citrated whole blood injected into the joint at the time of needle puncture (WT + Blood, ▲). Separate groups of mice were euthanized for histopathologic examination of hematoxylin and eosin–stained joints at each time point during the 8-week time course. Hemophilic synovitis was graded 0 to 10 points for increasing pathology, according to the Valentino mouse hemophilic synovitis scale,²⁵  which awards 0 to 10 points for increasing evidence of synovial overgrowth, neovascularity, and articular bleeding. Areas of greatest synovial thickening and vascularity were identified, and the average synovitis score from 3 nonoverlapping high-magnification fields was recorded. Points that are significantly different from “WT + Blood” are indicated by “*.” Points that are significantly different from “WT + NS” are indicated by “#.” Markers of significance are used as follows: *P < .05, ***P < .001, ****P < .0001, ^#P < .05, ^##P < .01, ^####P < .0001. (C) Time course of total chondrocyte number. Total chondrocytes were counted in a blinded manner in 10 randomly selected hematoxylin and eosin–stained fields of femoral and tibial cartilage using a 20× objective lens. The mean and standard deviation for the entire cohort of animals at each time point is plotted. Points that are significantly different from “WT + Blood” are indicated by “*.” Points that are significantly different from “WT + NS” are indicated by “#.” Markers of significance are used as follows: *P < .05, **P < .01, ^#P < .05. (D) Time course of apoptotic chondrocytes. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) using an In Situ Cell Death Detection Kit (Roche Diagnostics Corporation, Indianapolis, IN). TUNEL-positive cells were counted in a blinded manner in 10 randomly selected fields using a 20× objective lens. Points that are significantly different from “WT + Blood” are indicated by “*.” Points that are significantly different from “WT + NS” are indicated by “#.” Markers of significance are used as follows: **P < .01, ****P < .0001, ^###P < .001, ^####P < .0001.